OLIGOSACCHARIDE STRUCTURE & FUNCTION IN RECOGNITION

描述(由申请人提供)：在这笔赠款的30年期间，该项目为理解碳水化合物结构对糖蛋白的作用奠定了广泛的基础。在这段时间里，我们的研究确定了1)独特的寡糖结构作为特定血浆糖蛋白上的信号，2)碳水化合物特异性内胞受体识别这些结构的机制，以及3)这种识别在体内的重要生物学后果。我们已经从在分子和细胞水平上确定这些受体识别低聚糖的生物学基础，到在整个生物体水平上描绘这种识别的功能含义。我们在碳水化合物特异性内胞受体识别系统如何从循环中清除特定糖蛋白方面的进展现在达到了一个独特的机会，将这些研究带入了一个新的功能视角水平。我们已经通过生化和遗传学手段证明了甘露糖/半乳糖-4-硫酸盐受体(M/G4S-R)和去唾液酸糖蛋白受体(ASGP-R)分别介导了糖蛋白的清除，这些糖蛋白带有末端GalNAc 4SO4和末端Sia12，6GalNAc的N-连接寡糖特征。我们已经证明ASGP-R可以结合含Sia12，6-Gal的糖蛋白。我们推测，含有Sia12，6Gal末端的糖蛋白实际上是ASGP-R的主要内源性配体，并且ASGP-R是许多具有Sia12，6Gal末端的血浆糖蛋白浓度的关键调节因子。初步研究支持这一拟议功能。我们的发现为该受体提供了一个重要的新功能。我们现在将使用遗传模型和最近建立的蛋白质组学方法来确定ASGP-R和M/G4S-R的相对作用。我们将确定这些受体如何单独和联合作用，在生理和病理条件下，根据其碳水化合物特征调节血浆糖蛋白水平，从垂体糖蛋白激素调节雌激素的产生到对脓毒症的急性期反应。这些研究利用单受体和双受体敲除小鼠1)通过识别糖类信号来研究如何平衡生产和清除以调节血浆糖蛋白水平，2)确定扰乱这种平衡是否对生物过程有影响，以及3)建立产生具有不同碳水化合物特征的M/G4S-R亚型的分子基础，并确定这种受体亚型对生殖生物学的影响。我们揭示的基本机制信息最终将产生有助于评估诊断和治疗目的血浆蛋白变化的见解。 与公共健康相关：血液中的糖蛋白水平反映了它们的合成和去除之间的平衡。我们已经确定了肝脏中两个主要的碳水化合物特异性受体系统，它们通过确定糖蛋白的去除率来帮助控制血液中许多糖蛋白的浓度。我们将定义这些受体如何控制怀孕期间激素和炎性蛋白的浓度以及对损伤的反应。

DESCRIPTION (provided by applicant): Over the 30 years' duration of this grant, this project has established a broad foundation for understanding the role of carbohydrate structures on glycoproteins. In this time our studies have defined 1) unique oligosaccharide structures as signatures found on specific plasma glycoproteins, 2) the mechanisms of recognition of these structures by carbohydrate-specific endocytic receptors, and 3) the functionally important in vivo biological consequences of this recognition. We have proceeded from determining at the molecular and cellular level the biologic basis of oligosaccharide recognition by these receptors, to delineating at the whole organism level the functional implications of such recognition. Our progress on how carbohydrate-specific endocytic receptor recognition systems clear particular glycoproteins from the circulation now culminates in a unique opportunity to take these studies to a new level of functional perspective. We have demonstrated by biochemical and genetic means that the Mannose/GalNAc-4-SO4-receptor (M/G4S-R) and the asialoglycoprotein-receptor (ASGP-R) mediate the respective clearance of glycoproteins that bear the N-linked oligosaccharide signatures of terminal GalNAc 4 SO4 and of terminal Sia12,6GalNAc. We have shown that the ASGP-R can bind glycoproteins bearing Sia12,6Gal. We hypothesize that glycoproteins bearing terminal Sia12,6Gal are in fact the major endogenous ligands for the ASGP-R and that the ASGP-R is a key regulator of the concentration of numerous plasma glycoproteins that have Sia12,6Gal termini. Preliminary studies support this proposed function. Our findings give a significant new functional identity to this receptor. We will now use genetic models and recently established proteomic methods to define the relative roles of the ASGP-R and M/G4S-R. We will determine how these receptors act, individually and in combination, to regulate levels of plasma glycoproteins based on their carbohydrate signatures, under physiologic as well as pathologic conditions ranging from regulation of estrogen production by pituitary glycoprotein hormones to the acute phase response to sepsis. These proposed studies utilize single and double receptor knockout mice 1) to address how production is balanced with clearance to regulate plasma levels of glycoproteins through recognition of their carbohydrate signatures, 2) to determine if perturbing this balance has an impact on biologic processes, and 3) to establish the molecular basis for generating isoforms of the M/G4S-R with differing carbohydrate signature specificities and determine the impact of such receptor isoforms on reproductive biology. The fundamental mechanistic information that we uncover will ultimately yield insights that can aid in evaluating changes in plasma proteins for both diagnostic and therapeutic purposes. Public Health Relevance: The levels of glycoproteins in the blood reflect a balance between their synthesis and removal. We have identified two major carbohydrate specific receptor systems in liver that help control the concentrations of many glycoproteins in the blood by determining their rates of removal. We will define how these receptors control the concentrations of hormones and inflammatory proteins during pregnancy and the response to injury.